HEK293T cells were grown in Dulbecco’s modified eagle medium (DMEM, Gibco) in 10% FBS (Gibco), Penicillin (100 u/ml) (Gibco), and Streptomycin (100 μg/ml) (Gibco). HEK293T cells with the pSBtet-GADD34Δ/K3L construct inserted in the genome (ECE HEK293T cells) were grown in DMEM media (Gibco) supplemented with 10% Tet-system approved FBS (Gibco), Penicillin (100 u/ml) (Gibco), Streptomycin (100 μg/ml) (Gibco), and 2 mM GlutaMax (Gibco). Cells were grown at 37 °C in 5% carbon dioxide and 100% humidity. Induction of GADD34Δ and K3L expression prior to cell extract preparation is described below.
Tet system approved fbs
Manufactured by Thermo Fisher Scientific
Sourced in China
Tet-system approved FBS is a specialized fetal bovine serum (FBS) designed for use in cell culture systems that utilize the tetracycline-inducible (Tet) gene expression system. It is certified to be compatible with the Tet system, ensuring reliable and consistent performance in Tet-regulated cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Dmem media, by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute 1640 medium, by Thermo Fisher Scientific (1 mentions)
Cov362, by American Type Culture Collection (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
E0771, by American Type Culture Collection (1 mentions)
E0771, by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Tet plko puro, by Addgene (1 mentions)
Lipo3000, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Merck Group (1 mentions)
4 protocols using tet system approved fbs
1
Inducible Protein Expression in HEK293T Cells
2
Culturing Human Cancer Cell Lines
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Human osteosarcoma cell lines U2OS (ATCC) and COV362 (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% foetal bovine serum (FBS; Cytiva) and 1% penicillin–streptomycin (10 000 U/ml; Gibco). High-grade serous ovarian cancer cell lines OVCAR3, OVCAR8 (NCI Tumor Repository, Frederick, MD) and KURAMOCHI (Japanese Collection of Research Bioresources Cell Bank) were cultured in Roswell Park Memorial Institute 1640 medium (Gibco) supplemented with 10% FBS and 1% penicillin–streptomycin. BJ fibroblast HRAS(G12V) Tet ON cells were cultured in DMEM (Gibco) supplemented with 10% Tet-System Approved FBS (Gibco) and 1% penicillin–streptomycin (10 000 U/ml; Gibco). HRAS(G12V) expression was induced by 2 μg/ml of doxycycline. All cells were cultured at 37°C with 5% CO2 and checked for mycoplasma infection regularly.
3
Establishment and Characterization of Murine Mammary Cancer Cell Lines
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Murine mammary cancer cell lines EMT6 and E0771 were obtained from ATCC. Cell lines were tested at least once quarterly for Mycoplasma contamination. All media components were purchased from commercial vendors and prepared/stored under sterile conditions. Cell lines are utilized within the early passage (<30 passages from acquisition from ATCC) and are DNA fingerprinted through commercial services for validation. EMT6 cells were grown in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS; Life Technologies). E0771 cells were grown in RPMI (Gibco) and supplemented with 10% FBS. B2M-KO-IFNγ EMT6 cells were maintained in DMEM-F12 (GIBCO) supplemented with 10% TET-System approved FBS (GIBCO). B2M and Qa-1 knockout cell lines were generated using CRISPR plasmids (Santa Cruz) and flow sorted on GFP-positive cells. Cell lines were verified to be complete knockout populations by flow cytometry.
4
Overexpression and Knockdown of DKK1 in Osteoprogenitor Cells
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For DKK1 overexpression, human osteoprogenitor cells were transfected with DKK1 (HG10170-CY) and an empty plasmid (CV013) using Lipo3000 (Thermo Fisher, MA, USA) for 48 h. These plasmids were purchased from Sino Biological (Wayne, Beijing, china).
To construct the DKK1 knockdown cells, SaOS2 cells were cultured in RPMI 1640 (Hyclone) medium containing 10% Tet-System Approved FBS (Gibco) and 1% P/S. Cells were seeded in a 6 cm culture dish and transfected with shRNA vectors using Lipo3000 (Thermo Fisher) for 48 h. Transfected SaOS2 cells were selected with 1 μg/ml of puromycin (Sigma-Aldrich) and treated with doxycycline (Sigma-Aldrich) to induce knockdown of DKK1.
The vector sequences for knockdown of DKK1 expression were as follows: Empty: tet-pLKO-puro (Addgene), shDKK1: tet-pLKO-puro with the targeting sequence 5’-CCGG-AATGG TCTGGTACTTATTCCC-CTCGAG-GGGAATAAGTACCAGACCATT-TTTTTG-3’. Vectors were cloned by Cosmogenetech (Seoul).
To construct the DKK1 knockdown cells, SaOS2 cells were cultured in RPMI 1640 (Hyclone) medium containing 10% Tet-System Approved FBS (Gibco) and 1% P/S. Cells were seeded in a 6 cm culture dish and transfected with shRNA vectors using Lipo3000 (Thermo Fisher) for 48 h. Transfected SaOS2 cells were selected with 1 μg/ml of puromycin (Sigma-Aldrich) and treated with doxycycline (Sigma-Aldrich) to induce knockdown of DKK1.
The vector sequences for knockdown of DKK1 expression were as follows: Empty: tet-pLKO-puro (Addgene), shDKK1: tet-pLKO-puro with the targeting sequence 5’-CCGG-AATGG TCTGGTACTTATTCCC-CTCGAG-GGGAATAAGTACCAGACCATT-TTTTTG-3’. Vectors were cloned by Cosmogenetech (Seoul).
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